The tumor necrosis factor receptor (TNFR) family consists of a number of integral membrane glycoprotein receptors many of which, in conjunction with their respective ligands, are believed to regulate interactions between different hematopoietic cell lineages (Smith et al., The TNF Receptor Superfamily of Cellular and Viral Proteins: Activation, Costimulation and Death, 76:959-62, 1994; Cosman, Stem Cells 12:440-55, 1994). However, systemic expression of several members of this family suggest that these receptors may also play more general roles in organism development, homeostatsis, tumorigenesis, transplant rejection, septic shock, viral replication, and bone resorption. (Aggarwal, Nat. Rev. Immunol. 3:745-56, 2003).
The TNF receptor family is composed primarily of type I integral membrane glycoproteins which exhibit sequence homology, particularly with respect to cysteine-rich repeats in their extracellular domains. The TNF receptor family includes over 29 members (reviewed in Bodmer et al. TRENDS in Biochem. Sci. 27:19-26, 2002). A subgroup of this family whose members have particular structural similarities includes BAFF-R (Thompson et al., Science 293:2108-2111, 2001), BCMA (Gross et al., Nature 404, 995-9, 2000), TWEAKR (Wiley et al., Immunity 15: 837-46, 2001), EDAR (Monreal et al., Nat. Gen. 22:315-6, 1999), XEDAR (Yan et al., 290:523-7), TACI (von Bülow and Bram, Science 278:138-141, 1997), and MK61 (Theill et al., WO0220762, 14 Mar. 2002), This group of TNFRs are distinguished by having two or fewer cysteine-rich domains along with a more variable cysteine-rich pattern than what is commonly seen.
In general, members of the TNF receptor family are characterized by a multi-domain structure comprising an extracellular region, a transmembrane domain, a linker region between the extracellular ligand-binding region and the transmembrane domain, and a cytoplasmic domain. In several members of this family (TNFR 1, Fas, DR3, DR4, DR5, DR6, NGFR and EDAR) this cytoplasmic domain contains a death domain associated with apoptosis. These members of the TNFR family, as well as others that do not include death domains such as TACI and BCMA, have been shown to bind one or more of the six tumor necrosis factor receptor-associated factors (TRAF1-6). These factors bind to the intracellular domain of the receptor at a short consensus sequence and act to couple the receptor to internal cell signaling pathways.
The extracellular ligand-binding region is characterized by the presence of one to six cysteine-rich motifs each containing about six cysteines and approximately 40 amino acids, although variation in the size and number of these motifs occurs among members of this family. The cysteine-rich regions provide the motif for binding to shared structures in the ligands. The highest degree of homology among the TNFR family members is within this extracellular cysteine-rich region. Among human TNFRs the average homology is in the range of 25% to 30%. Between the last cysteine-rich repeat and the transmembrane domain is a small spacer region of between 8 to 70 amino acid residues. Cell surface TNF receptors are anchored in the cell membrane by a transmembrane domain characterized by a sequence of hydrophobic amino acid residues. On the opposite end of the protein from the extracellular ligand-binding region and separated from it by the transmembrane domain is the cytoplasmic domain. The cytoplasmic domains of TNFR family members are small, from 46 to 221 amino acid residues, which suggests possible differences in the signaling mechanisms among family members. In the TNF receptor for example, activation is triggered by the aggregation of cytoplasmic domains of three receptors when their corresponding extracellular domains bind to trimeric ligand, which may be a common method of activation for the receptor family.
One member of the TNF receptor family, osteoprotegerin (Simonet et al., ibid.), is unique in that it is a secreted protein. Soluble forms of other TNF receptors have been described for TNFR-I, TNFR-II, low-affinity NGFR, FAS, CD27, CD30, CD40 and 4-1BB, but these were generated either by cleaving from the cell membrane or secreted by alternatively spliced mRNA. OPG inhibits osteoclast maturation and it is thought that it might serve to regulate bone density by modulating osteoclast differentiation from hematopoietic precursors. OPG provided protection from normal osteoclast remodeling and ovariectomy-associated bone loss.
The X-ray crystallographic structures have been resolved for human TNF (Jones et al., Nature 338:225-28, 1989), LT-a (Eck et al., J. Biol. Chem. 267:2119-122, 1992) and the LT-a/TNFR complex (Banner et al., Cell 73:431-45, 1993). This complex features three receptor molecules bound symmetrically to one LT-a trimer. A model of trimeric ligand binding through receptor oligomerization has been proposed to initiate signal transduction pathways. The identification of biological activity of several TNF members has been facilitated through use of monoclonal antibodies specific for the corresponding receptor. These monoclonal antibodies tend to be stimulatory when immobilized and antagonistic in soluble form. This is further evidence that receptor crosslinking is a prerequisite for signal transduction in this receptor family. Importantly, the use of receptor-specific monoclonal antibodies or soluble receptors in the form of multimeric Ig fusion proteins has been useful in determining biological function in vitro and in vivo for several family members. Soluble receptor-Ig fusion proteins have been used successfully in the cloning of the cell surface ligands corresponding to the CD40, CD30, CD27, 4-1BB and Fas receptors.
Ligands for these receptors have been identified, and with one exception (NGF) belong to the TNF ligand family. The members of the TNF ligand family share approximately 20% sequence identity in the extracellular ligand-binding regions, and exist mainly as type II membrane glycoproteins, biologically active as trimeric or multimeric complexes. Although most ligands are synthesized as membrane-bound proteins, soluble forms can be generated by limited proteolysis. For some receptors, solublization is necessary for activity, while for others, their activity is inhibited upon cleavage.